N = 28) and after  the addition of the balcony. Locomotor and morphological differences were compared after a mild-moderate T9 spinal cord contusion injury in wild-type mice. Post-injury assessments of locomotor function for 6 weeks included Basso Mouse Scale (BMS) and treadmill kinematic assessments (week 6). Balcony-housed mice showed greater improvements not only in basic locomotor functions  compared to those in standard housing, but also surpassed mice in standard housing without the balcony in higher-order locomotor recovery outcomes, including BMS late-stage recovery measures . Additionally, balcony-housed mice had overall higher BMS scores, consistently attained more BMS subscores, and had better treadmill track width and stride length compared to those with no balcony. The housing enhancement of a balcony led to unforeseen consequences and unexpected higher recovery outcomes compared to mice in standard housing. This retrospective study highlights the importance of housing conditions in the key outcomes of locomotor recovery after incomplete contusive SCIs in mice.It is well established that both positive and negative housing conditions of laboratory animals can affect behavioral, biochemical, and physiological responses. Housing enhancements have been shown to have beneficial effects on locomotor outcomes in rodents with spinal cord injury (SCI). Subsequent to an unplanned housing enhancement of the addition of a balcony to home cages by animal care personnel at a research facility, a retrospective analysis of multiple SCI studies was performed to determine whether outcomes differed before (four studies,  However, unanticipated events can result in unintentional effects on recovery outcomes.4–6A variety of rehabilitative methods have been used to promote functional locomotor recovery after spinal cord injury (SCI) in the rodent model, including activity, exercise, and step training with treadmill walking.During a series of SCI research experiments at a research facility, the standard housing conditions of the laboratory animals were altered by the animal care facility, inadvertently changing the housing conditions of subsequent experiments compared to earlier identical studies. A balcony was added to the home cage of uninjured and mid-thoracic SCI mice to enhance well-being. After multiple experiments, it appeared that mice in the home cage with the balcony had better locomotor recovery post-injury compared to mice that had been housed in standard cages.8 Studies using environmental enrichment that include in-cage enhancements by inserting objects to encourage activities, such as tunneling, incline walking, and climbing behaviors, have shown improvements in locomotor recovery.10 Moreover, there is evidence that acute spontaneous effects referred to as “self-training” or “in-cage training” can have positive results on functional recovery.11–13 Housing enhancements as well as spontaneous self-training can unintentionally influence experimental research outcomes as a result of increased activity.5 Other environmentally related housing factors, including housing density, cage size, and design, can lead to experimental outcome variability and affect scientific validity.6 The following retrospective analysis was undertaken to determine whether an unintended housing enhancement during a series of SCI research experiments resulted in unexpected enhanced locomotor recovery in mild-moderate SCI in mice.It is well established that the well-being of laboratory animals, as well as their behavioral, biochemical, and physiological responses, can be affected by housing conditions.N = 11, B: N = 5, C: N = 2, and D: N = 10; total = 28) with four studies where a balcony was added to the home cage . These eight experiments included four published14 and four yet-to-be published studies.A retrospective analysis was performed to compare four studies where mice were housed in standard cages  mice . Mice read libitum. In four experiments, a balcony was inserted in their home cages (7 in length × 5 in weight × 2¾ in depth) with easy accessibility for climbing (Mice were group housed in standard cages (3–5 per cage) for 6 weeks post-injury. Food and water were available climbing  through 18 All components of BMS scoring were examined  by two trained observers blinded to experimental conditions.examined , “Added”20 Briefly, a high-speed digital video camera recorded the ventral view of the treadmill belt as animals walked across. Images were recorded at 100 frames/s and analyzed using MaxTRAQ . Acceptable passes included walking in the middle of the treadmill, stepping with few lateral deviations, and stepping without pauses.Treadmill walking was assessed , as previously described.17 To detect the extent of spared white matter (SWM) at the injury epicenter, iron eriochrome cyanine with an alkali differentiator was used to stain myelin, as previously described.22 Sections were imaged, quantified, and the epicenter identified, as previously described.17 SWM from the experimental group was normalized to average white matter content at identical spinal levels from a separate cohort of naïve control animals.At 6 weeks post-SCI, mice were perfused and spinal cords processed, sectioned, and mounted, as previously described.23 TreadScan assessments were normalized to baseline (BL) values (week 6/BL × 100) and analyzed using repeated-measures analysis of variance , followed by Bonferroni post hoc t-tests. Independent t-tests  were limited to reduce the probability of a type 1 error and were used to compare the balcony groups' kinematic speed and coordination measures.24 Non-parametric Spearman rank correlations were performed among BMS, TreadScan, and SWM outcomes.23 Values represent mean ± SD. Data were analyzed using SPSS statistical software .Four BMS outliers (≥2.5 standard deviations [SDs]) were removed. BMS scores and component left- and right-side values were not different and were averaged. Time course of recovery was assessed by averaging acute (days 3 and 7), chronic (weeks 5 and 6), and all  time points. Group comparisons of BMS component scores were performed using non-parametric Mann-Whitney U tests. Frequency counts were compared using binomial proportion tests, corrected for small sample size when appropriate.Balcony group mice had higher BMS , BMS + subscore  compared to the group without a balcony  when walking on the treadmill compared to those without a balcony , as well as the Plantar Stepping Index (a measure of forelimb to hindlimb stepping), did not differ between the balcony groups .Terminal assessment of the amount of SWM 6 weeks post-injury yielded no significant group difference  is a fundamental component of functional recovery, it is likely that rehabilitative training incorporating BWS alone is not sufficient to restore locomotor function beyond what occurs “spontaneously.”3 Injured animals undergoing rehabilitative activities, such as step training with BWS, either through a harness and treadmill or in shallow water, have the ability to produce well-organized patterns of stepping on a treadmill. However, they are unable to do so overground because of a lack of balance.29–31A key component necessary to improve functional locomotor recovery and enhance plasticity begins with weight-supported stepping.32–34 The modulation of muscle output by sensory inputs leads to “fine-tuning” in which the basic locomotor pattern generated by the central pattern generator is fine-tuned to adapt to terrain and walking surface changes.36 This fine-tuning is dependent on the step phase (stance vs. swing) and thus is important for both intra- and interlimb coordination.37Locomotor training techniques have revealed the need not only for load-bearing weight support for functional improvement, but also the importance of proprioceptive input.The outcome measures of the BMS, its components, and kinematic assessments utilized to assess locomotor functional recovery in the present studies can be categorized according to basic  and higher-order  functional outcomes. In the present studies, balcony group mice showed greater improvement in the BMS's early stages of recovery for basic ankle limb movements, plantar placement, and plantar stepping, in addition to kinematic foot base of support. Thus, mice with the balcony improved more in all basic locomotor recovery abilities compared to mice without a balcony, first developing weight support, then plantar placement of the paw, followed by weight-supported plantar stepping.As previously alluded to, the relationship between trunk control and balance is an integral feature in the successful acquisition of basic locomotor function after SCI. Moreover, it also plays an important role in the subsequent improvement of more complex higher-order locomotor abilities. Mice housed with the balcony performed better in multiple balance-related outcome measures, including paw rotation, trunk stability, and tail position, with a corresponding reduction in adverse trunk events. Also, kinematic outcomes for stride length and track width were better for balcony-housed mice compared to those mice housed in standard cages. These achievements illustrate that mice housed with access to the balcony advanced beyond basic weight-supported stepping, exhibiting continued improvements in stability and balance over and above those of standard housed mice.The progression to fine-tuned complex motor behaviors representing higher-order recovery also was better in balcony-housed mice compared to those housed without the balcony. This is exemplified by balcony group mice having higher overall BMS and BMS + subscore results, clearly demonstrating better overall locomotor performance. In addition, other evidence of higher-order functional outcome measures supporting greater improvements in mice housed with a balcony compared to those in standard housing include the significant relationship among locomotor and kinematic functional outcomes. Specifically, there was a positive relationship between BMS subscores and kinematic rear track width for balcony-housed mice only, suggesting that the continued improvement in stepping and finer details of locomotion were related to recovery of balance.However, neither BMS nor TreadScan outcome measures for coordination revealed significant differences between the balcony and no-balcony groups. It is suggested that climbing up onto the balcony and other activities promoted by the presence of the balcony likely contributed to the ability for plantar placement of the hindlimbs. In turn, it is conceivable that an increase in basic plantar placement was associated with increased muscle strength attributable to repeated limb-loading at acute time points with or without improvement in balance.Another important indicator supporting functional improvement can be gleaned from the morphological assessment of SWM. In the current studies, direct comparisons of the SWM area between groups revealed no significant differences; however, the relationship between SWM and stride length were different for the two groups. Animals without the balcony had poor stride lengths while walking on the treadmill, and poor stride lengths were significantly related to poorer white matter sparing outcomes. Taken together, these assessments, along with the morphological findings, confirm the superiority of fine-tuned locomotor skills for mice with access to the balcony.1 A common goal of rehabilitative methods that utilize training is the development of a skill that is retained post-training. It could be argued that the addition of the balcony resulted in increased exercise through the activity of climbing and constituted the attainment and practice of an acquired skill without formal or applied training. Although activity levels were not directly measured in the current studies, climbing is a natural behavior in mice and, given the opportunity, they will spend a considerable amount of time engaging in high-energy activities, such as climbing and wheel running, compared to walking or other natural  behaviors.39 As concluded by Büttner, “climbing is obviously a regular component of activity…and is a factor in locomotor activity.”39Adkins and colleagues promoted the concept that different skills result in different physiological changes and that the properties of strength, endurance, and skill training are necessary components to accomplish locomotor improvement.40 Thus, given the opportunity, animals with the capability will engage in both their “natural” (climbing) and “un-natural” (wheel-running) behaviors if present and available in their environment.41In the present studies, where level and severity of the SCI did eliminate the ability to climb acutely post-injury in all animals, it is likely that balcony-housed mice had increased exercise by engaging in climbing. This is supported by a study using rats with mild-moderate SCI housed in environmentally enriched cages with activities for climbing that reported significant improvement in locomotor scores and subscores, as well as the kinematic outcome measures, thus attaining higher-order fine-motor skills as compared to animals housed without enrichment.39A higher percentage of balcony-housed mice were able to achieve frequent stepping compared to those without the balcony , suggesting that the activities made possible by the balcony  influenced the capacity to step. Thus, the specific preference for climbing compared to other activities demonstrated previously support the contention that mice in the present studies were highly likely to engage in the activity of climbing, even though this behavior was not monitored.1 Moreover, climbing likely involves most of the five levels of motor control needed for locomotor recovery as defined by Prochazka and Yakovenko: skeletomuscular and stretch reflex properties associated with load compensation, body motion and pattern generation, adaptation and prediction, and behavioral goals and context.33The improved outcomes reported here suggest that the activity of climbing positively influenced at least two of the three training-related tasks as outlined by Adkins and colleagues: skill and strength, and possibly the third, endurance for locomotor recovery.The present studies show a clear improvement in locomotor recovery in SCI mice after exposure to a housing enhancement , adopted primarily to improve animal well-being and reduce stress. The incorporation of a balcony in the present studies represents both a type of environmental enrichment with increased activity levels and also, simultaneously, the opportunity for in-cage self-training that involves whole-body movements, trunk control, limb strength, and balance. Results support the contention that the addition of a balcony provided training opportunities that allowed critical milestones in basic and fine-motor skills to be acquired that surpassed those of animals in standard cages without a balcony. These studies reveal that substantial functional locomotor improvements can occur unintentionally from enhanced housing that encourages an increase in general activity and specific skill-learning, such as climbing.The capability of experimental studies to discern effects from experimental manipulations versus extraneous, unintended influences is imperative for scientific validity. In the current body of studies, the unplanned addition of a balcony to standard cage housing resulted in unintended effects to experimental outcomes in SCI mice. Results demonstrated broad functional improvement, encompassing both basic and higher-order locomotor abilities in mice housed with the balcony compared to no balcony. The present results clearly illustrate that significant functional changes in locomotor outcomes can occur unintentionally after mild-moderate contusion SCI from enhanced housing that encourages an increase in general and specific activity such as climbing.


N = 12) talk about their identities, prosocial behaviors, and connections between them. Of special interest was whether and how the participants included their experiences of dehumanization. Focus group data were analyzed using modified analytic induction. Participants felt good about their racially gendered identities but felt they occupied a precarious position in the United States. Participants’ beliefs about how others viewed them motivated restraint from engaging in too many prosocial acts to prevent appearing vulnerable. Participants explicitly referred to their experience of oppression in these discussions and its interaction with identity and prosociality. Results suggest research must consider how macro‐level processes like racism influence the identities and prosocial behaviors of adolescent Black males.This qualitative study examined how adolescent Black males ( During adolescence, Black males in the United States face complex and daunting challenges Brooms, . In K‐12Despite growing up in the context of anti‐Blackness, many Black youth develop positive racial‐ethnic identities (REI); these youth have higher self‐esteem, better short‐ and long‐term academic outcomes, and are less affected by discrimination than peers who have more negative feelings about their REI , and they contribute to academic and social success, two important outcomes of adolescent development  to control their bodies Coates, . The higperceptions of their macro‐ and micro‐social contexts  is an identity‐centered ecological model that focuses on an individual’s Spencer, , 2008. Racross contexts. Net vulnerability balances possible risk factors  with possible protective factors (supportive parents and strong community ties) to determine overall risk. Similarly, net‐stress engagement balances an adolescent’s actual engagement with stress with their reactive coping mechanisms. The adolescent’s net engagement with stress will lead to positive or negative outcomes that are represented within the stage‐specific outcome component. We focus here on three components of P‐VEST: net vulnerability, emergent identities, and stage‐specific outcomes. We foreground anti‐Blackness as a contributor to net vulnerability and examine whether and how adolescent Black males speak about their vulnerability in relation to their emergent identities and to the stage‐specific outcome of prosociality.Phenomenological variant of ecological systems theory is comprised of five bidirectional components: emergent identity, net vulnerability, net stress engagement level, reactive coping mechanisms, and stage‐specific outcomes Spencer, , 2008. AIntersectionality theory describes the ways interlocking systems of oppression shape one’s experience  investigation to examine if and how adolescent Black males’ racial‐ethnic and gender identities predict self‐reported prosocial behaviors within an urban school context  identified as male and as Black or African American. The sample size constitutes all available participants for the qualitative portion of the larger mixed‐method study. The mean of prosocial scores on the Prosocial Behavior Scale  was 3.04. The participants invited to participate in the qualitative portion scored in the top and bottom quartile of prosocial scores and represented over 50% of their group’s respective quartile, supporting saturation efforts  (a pseudonym), a public charter high school operating within a large metropolitan school district in the southeastern region of the United States. Over 98% of students attending CA identify as Black or African American. College for All is a Title 1 school  located in an economically depressed area. Additionally, all students attending CA qualified for the federal lunch subsidy, indicating their family incomes were at or below 130% of the national poverty level.The IRB at Georgia State University approved the present study (IRB00000716). All invited participants elected to participate in a focus group. They were provided a pizza lunch and 10 dollars for their participation. Focus groups were conducted on school grounds in Fall 2017. Each session took approximately 65 min, and both were audio‐recorded. See Appendix between participants is equally important to information told directly to the researcher  and the contextual variable perceptions of discrimination were used as initial descriptive codes and were applied line by line to the transcripts. Any lines containing irrelevant information (such as discussing favorite pizza toppings) were excluded from coding.We used modified analytic induction when coding qualitative data , more specific interpretive codes were added. A line such as “I bring her her favorite snack” was further coded with the interpretive code Data were coded with an educational psychology doctoral student using the mixed method coding software Dedoose  can be spaces of liberation rather than contexts of oppression. She believes that we must listen to the voices of Black youth to best understand their development and resistance to the anti‐Blackness endemic to the U.S. context to achieve this and therefore uses research methodology that centers participants’ perspectives. The second author is a White female senior faculty member in a large urban research university in the Southeastern US. During the last 15 years her research has focused on Black adolescents’ girls’ thoughts and feelings related to psychological development within urban school contexts. The first author conducted focus groups and led analysis of the data.The positionality of the authors informed the research methodology. Given the authors positionality as women, it was particularly important to utilize methodological approach that centered the voices of participants, adolescent Black males. Through modified analytic induction and focus groups, participants’ beliefs were the guiding point for focus group questions and follow up questions. Moreover, the subsequent analysis used a lens that centered participants’ race and gender through a macro‐level lens to understand their experiences.Participants described their feelings about their gender identity, REI, and racially gendered identity. In both groups the words they used to describe their gender were: “powerful,” “trustworthy,” “strong and smart,” and “leader.” For race, participants said “scared,” “discriminated,” “unique,” “targeted,” “humble,” “powerful,” “blessed,” “talented,” “highly favored,” and “feared.” When asked about what words come to mind regarding their racially gendered identity as adolescent Black men, the following descriptions emerged: “fear,” “oppressed,” “flashy,” and “provider.”We asked participants if their perspectives represented their point of view as a Black person, a male, or a Black male. All participants stated their perspective was of a Black male. As Rico stated, the identities “come together.” Both groups agreed they could not untangle gender from race. Interestingly, despite stating the two identities were intertwined, the conversations in the groups focused primarily on REI.Yeah, I was going to say, I feel good about being Black because it just—I feel like I’m bonded with everybody. There’ll be people I don’t even know. I can just be like ‘Hey, how you doing?” or “What’s up?’Participants expressed high private regard for their REI. In both focus groups, participants discussed that despite Black Americans facing a range of historical and present‐day traumas, they thrive and maintain a strong sense of community. Some went further and cited Black Americans’ resilience as evidence of “Black people being blessed” and a source of pride for them. Tyrese extended this sentiment.Because you can see, in everything we do, we already got some hate thrown at us. Like most of the movies and shows on TV, it’s White folks on there. Then once you go into the suburbs—cuz they separate it, the suburbs, then urban. All the Black folks in the urban with all the broke down stuff. Messed up TVs, all that. Then you got the suburbs with the White folk.Participants’ responses on how other people viewed their race  were mainly negative. They were keenly aware of the discrimination Black Americans faced. Zaire from the low prosocial group used the following example of segregated communities and media portrayals of Black Americans to illustrate this point.…I say feared…cuz I know I’ve been walking down the street before and a cop just looked at me and approached me for—and talked to me, stuff like that. I’ve been in places where White people see us, and they clutch their purses. They look at us the wrong way, go to the opposite side of the way.While he spoke, other participants nodded their heads, quietly affirming his frustrations about the persistent inequality in America. Participants in both groups used words like “dangerous,” “feared,” and “targeted” when describing other peoples’ feelings and opinions about Black people and Black males. When probed on the fear aspect, Chad from the high prosocial group had this to say:They pitch to us, like when we leave high school, sports are gonna get us out. They don’t pitch us as doctors or lawyers or stuff like that. They try to teach us to dumb us down, so that—because the Black man in power is—he’s dangerous to everybody else… I think they only accept us when we’re making their money instead of takin’ away from their pockets. That’s when they try to get rid of us.They believed others viewed their racially gendered identity in a positive light only when related to certain stereotypes about Black men. In both groups, participants stated the two areas in which they were positively perceived by others were in sports and with women. To quote Rico in the high prosocial group, “They like us when it comes to our athletic abilities and sexual desires.” In the low prosocial group, Ray said,Regarding sex, the commentary moved into a back and forth about White girls approaching them, saying things such as, “We like Black boys.” Participants were not disturbed by these experiences but rather amused and a bit boastful. For example, when Michael from the high prosocial group proudly stated, “White girls love us,” all other participants vigorously agreed.treated a particular way because of their race. Most examples provided were of other people treating them with suspicion. Participants surmised it was due to their race or their positioning as Black males. Tyrese shared the following experience,…I was on the train. A couple players on my football team, we were just walking on a train. People were giving us looks. We were just chilling. Some people would like to move away. I don’t know why. I guess cause we were Black.Participants were then asked to think deeply about instances when they were they. When probed on whom they referred to, Maurice stated “White people” absentmindedly while playing with his pencil, and the others quickly nodded in agreement. When asked to elaborate, Maurice made eye contact and firmly stated, "Look at our presidentWhen asked to share specific experiences of being treated a certain way due to their identity, the low prosocial group recounted only examples of negative treatment. During their conversations and descriptions of events, low prosocial group members frequently used the term us.” Others in the low prosocial group stated learning about American history shaped their current perspective. Ray explained, “It started when like ever since I started learning about slavery… As I got older, as I started to notice history repeats itself. It gained on me. It got bigger.” Participants agreed learning more about the world and their positions as young Black males soured them to the intentions of White Americans.The group went on to state these opinions were formed as they “got bigger” with Ashad elaborating, “As I got bigger—I became a bigger target.” The justice system also influenced these views; Maurice quietly stated, “The White man have justice over they frequently but when probed further about it, participants stated “they” referred to anybody. Tyrese explained:I see it everybody the same. It’s just that’s what they see, what they perceive the world as and what you see it as may be different because so many people see the world in their own type of way.The high prosocial group presented a different perspective on the relationship between White and Black Americans. When asked to share experiences of positive or negative treatment predicated on race, only half of the group reported negative experiences they believed to be due to their race. This high prosocial group also used the word Some people, and I’m not even gonna label them as White people, just they cool people, I’m not gonna be like, ‘Oh that’s a White person over there.’ I’m trying to get past that. I still wouldn’t tolerate a White person calling me the n‐word tho.Chad expounded on Tyrese’s point, explaining:Participants in both groups stated that others’ feelings about Black Americans did not affect how the participants in turn behaved toward them. However, the low prosocial group was more explicit than the high prosocial group in naming and critiquing White people.To probe their thinking about prosociality in a conversational way, participants were first asked to provide examples of kindness . The examples were, by and large, acts of generosity. The two most common acts were sharing food or money with those around them. Responses included “Help with work and stuff,” “Kind is when you nice to everybody, and you just—you just not mean to nobody,” “Helping with priceless things. Help with work and stuff,” and “You just nice to everybody. Just a good person.” When further probed, participants agreed kindness was going beyond what was expected of you. Jared and Kenny in the high prosocial group discussed how they were kind to other students at their school. Jared said, “Just on a day‐to day‐basis, opening the door for anybody‐ just hold the door.” Kenny stated, “Matter afact, just earlier I just gave someone a dollar because he asked me for a dollar…if you need help, I got you.”because of respect for them. Participants defined respect as an understanding of physical and emotional boundaries. As Kenny succinctly put it, "Respect is people knowing what I tolerate and what I don't tolerate." Kindness, or even liking another person, was not required to give or receive respect. Bobby shared:Like, I don't like you, but I respect you as a man. You see someone getting picked on, or you see‐ if you see an incident going on…like a fight. The person he got a big presence and like, ‘Nah man I ain’t gonna fight you.’ I respect you. Don't mean we tight. That don't mean we cool, but I respect you for being a bigger person…I respect you as a man, but that don't mean we got any type of friendship, or anything. I just respect you.Besides generosity, respect was one of the first words participants in both groups used when asked to define being kind to another person. When asked to expand upon respect, participants stated being respectful was different from being kind or generous. According to participants, one is often prompted to engage in prosocial behaviors with another person Within their school context, participants showed respect to peers and school staff through acts of deference. As an example, participants cited acquiescing to a teacher’s demand without complaint or comment, even if they did not understand or agree with the request. Zion stated, “Respect is like if she tell you to be quiet, just be quiet. Don’t talk back.” Similarly, with peers, participants showed other students respect by not engaging in or escalating a potentially volatile situation. When asked about which they valued more, respect or kindness, all participants stated respect. Participants felt it was essential to be respected across contexts. They stressed a nice person who did not show them respect was essentially worthless.I don’t wanna be—I don’t wanna come out the hood doing good and not leavin’ them. It’s like, say I did come out, I make it, and then like, dang, he was over here and he ain’t even doin’ nothin’ for us. I wanna at least give back so they could have an idea like do right.Participants reflected on how their identity, the context of dehumanization, and their prosocial attitudes interact in their lives. They described how racial socialization influenced their identity and that their identity was influential in how they treated others. Some participants said they were raised to be kind, and Black people in general are community oriented. When asked how their REI motivated them to be kind to others, Tyrese and Tommy in the high prosocial group explained the relationship. Tyrese started by stating, “It’s in my heart,” and Tommy confirmed these sentiments in saying, “It’s just the way we were brought up.” The low‐prosocial group had similar beliefs, and Locksley from the group discussed the community aspect that was important to his identity.Zion: Okay…so that moment you walk by the car, and she lock the doors so you—that makes you wanna go harder so 10 years later you a successful Black man. She lookin at the TV. She see your name pop up and she say, ‘Oh that look exactly like the Black boy that walked by my car and I locked the door on him.’Ashad: I think if they lock the doors, the next day come say something to her. Say something nice to her. Don’t try to act aggressively and keep doing that. Then eventually, she’ll feel stupid for even trying to treat you like a threat or something.Although motivated to give back to their own community, the threat of being stereotyped in the larger world was simultaneously looming. To counter negative stereotypes about Black males being dangerous, some participants were motivated to be successful or kind to show the person the error of their ways. Zion and Ashad from the low prosocial group described a hypothetical situation with a woman who may lock her doors upon sighting a young Black male.The participants with the highest prosociality scores were explicit about the limits and risks of being kind, about needing to protect themselves from appearing too kind and thus being vulnerable. This was not observed in the low prosocial group.Kenny: Let’s say I come—we come to school every day and I give you a dollar every day. You be like thank you, thank you the first time I give you a dollar. If I constantly keep giving you that dollar you gonna expect me to did not giving you that dollar.Jared: Then that one day you don’t give them a dollar!Kenny: Then they are gonna get mad at you, ‘Where’s my dollar?!’ I’m like ‘Yo dollar?! What you mean?! I gave it out my heart, my kindness.When asked what they would tell a younger brother about being kind, Sammy in the high prosocial group responded, “I’d tell him to treat people the way you want to be treated. Don’t be easily provoked…Sometimes they make take your kindness as a weakness, but I feel like I should just treat people like you want to be treated.” In response to this, Kenny stated, “Be kind, but don’t be too kind,” and Malcolm added, “Don’t be vulnerable.” This began an exchange in which they discussed how they may be taken advantage of if they are too kind to those around them. Kenny and Jared used a hypothetical situation to illustrate their point.Kenny explained one consequence of their oppressed status, saying, “It’s being vulnerable because they feel like us being Black, we’re vulnerable to rap. They feel like if they provoke us enough, we’ll explode.” They went on to explain their identities as Black males made them more susceptible to being taken advantage of or unfairly tested. Malcolm stated, “The fact that we’re Black men, we’re the target anyways. They’re going to try to see if the stereotypes if they hear are true…Some folks in the world—they want to see you—I don’t know—they want to see you fail.”Participants stated there are only a few people with whom they can be vulnerable and kind. They named their mother as one with whom they were unabashedly open and generous. As one participant stated, "My mom, that's all I need in this world." They went on to state certain contexts afforded vulnerability such as a sports game or graduation, but generally, they were very protective of their feelings, which influenced the ways they engaged with others.This study examined adolescent Black males’ talk about their racially gendered identity and prosocial behaviors, the relationship of these constructs to each other, and the role of systemic bias in the meaning‐making process. Theory  shape emergent identities Spencer, . As predScholars have noted that Black males must develop an identity in a terrain in which they are simultaneously desired and despised Cooper, . As BoykA difference between the focus groups emerged in the participants’ interpretation of the forces at play that create hostility and discrimination in their environment. As found previously in the quantitative study, those participants who scored lower on a measure of prosociality also scored lower on racial public regard, or how they believe others see them. In their focus group the low prosocial participants recounted episodes of being treated negatively because of their social position, and they used “othering” language in their narratives, referring to those who mistreated them as “they.” When probed, the participants described “they” as Whites and painted a picture of their social world as a battlefield where White and Black Americans are continually at odds. The low level of prosocial behaviors they reported in their immediate social network could be a coping response to their perceived powerlessness in the face of discrimination. They also may be unwittingly incorporating society’s negative stereotypes about Black males into their burgeoning identities . These findings recall previous studies illustrating the contribution of racial socialization messages to prosociality  to protect themselves.The interaction of factors in these adolescent Black boys’ social development is illustrated in these findings. The participants described the complexities and contradictions they negotiate in constructing a self and moving through the world. For example, the participants feel pride about who they are and at the same time understand that the larger world is hostile because of who they are. They describe how their identity includes the values of kindness and respect, while others stereotype them as inherently callous and dangerous. To protect their identity and possibly their physical integrity, these youth go out of their way to be prosocial, to dispel negative stereotypes. At the same time, they guard their behavior and try not to be or because of the low regard others have for them. One may not think of “helping behaviors” as a coping mechanism but where adolescent Black males are considered threatening, prosociality creates a counternarrative for others and possibly also for themselves.These findings echo established predictors of prosociality in adolescent Black boys  identity in Black boys can promote prosociality (to cope with and rebut stereotypes) and community engagement (as an act of solidarity), but also can promote guardedness and withdrawal from engagement (to look imperturbable and deflect threat). The development of prosociality is frequently characterized in relation to micro‐level influences like peers and parents, but these findings indicate the macro‐level context of racial discrimination and inequity influences how adolescent Black males think about the social world and the meaning and purpose of prosociality. Our participants’ beliefs about the fundamental elements of social relationships were constructed through their understanding of themselves as Black males which was, in turn, informed by anti‐Blackness and systemic oppression within the United States.While this study uncovered valuable information about Black males’ social development, some limitations should be noted. Our small sample size and single location limit generalizability but allowed us to learn important detail about the participants’ thinking. We also were not able to look at differences by age or over time. A longitudinal study may reveal shifts in these processes. Additionally, grouping participants by prosocial scores may have led to the absence of more varied voices and exaggerated the group differences. Further, we realize social acceptability may have influenced participants’ responses in the focus groups. Last, we acknowledge that our data are based on boys’ quiet reflection in a safe space, and that may be different from their behavior in real time. Despite these limitations, this study demonstrates the value of using qualitative approaches, the P‐VEST framework, and an anti‐Blackness lens to understand pathways and outcomes for youth of color.One finding meriting future investigation was the importance of respect to these adolescents’ thinking about relationships. Future examination of the function and development of respect can elucidate its relationship to adaptive developmental outcomes. Similarly, reflected appraisals, particularly the influence of low racial public regard, were central to our participants’ thinking and merit further investigation to explicate the processes of development in a hostile context.These findings also demonstrated the ways adolescent Black males both accommodate and resist societal expectations of them. Future research should explore the risks and benefits for development that arise as marginalized youth manage dominant messages about who they should be and how they should act. Moreover, given the specific experiences attached to racially gendered identity in these findings, using an intersectional lens will be essential to further our understanding.because of their entrance into adolescence. Thus, future research on young Black males must include a developmental perspective that explicitly considers the role of macro‐levels processes like racism and discrimination in normative developmental outcomes.Finally, as previously noted, adolescent Black males must negotiate tensions between their private regard and public disregard during their adolescence


The skin, as the largest organ of human body, can use ions as information carriers to convert multiple external stimuli into biological potential signals. So far, artificial skin that can imitate the functionality of human skin has been extensively investigated. However, the demand for additional power, non-reusability and serious damage to the skin greatly limits applications. Here, we have developed a self-powered gradient hydrogel which has high temperature-triggered adhesion and room temperature-triggered easy separation characteristics. The self-powered gradient hydrogels are polymerized using 2-(dimethylamino) ethyl metharcylate (DMAEMA) and N-isopropylacrylamide (NIPAM) under unilateral UV irradiation. The prepared hydrogels achieve good adhesion at high temperature and detachment at a low temperature. In addition, according to the thickness-dependent potential of the gradient hydrogel, the hydrogels can also sense pressure changes. This strategy can inspire the design and manufacture of self-powered gradient hydrogel sensors, contributing to the development of complex intelligent artificial skin sensing systems in the future. As an important organ of the human body, the skin can not only protect the human body from external bacteria and viruses, but also accurately receive and sense various external physical and chemical stimuli. To date, people have developed multiple artificial skins to realize the function of human skin ,2,3. AmoTraditional artificial skin adhesives mainly include bionic microstructure adhesives inspired by octopuses ,17,18, tHere, we have developed self-powered gradient hydrogel sensors with a reversible adhension capacity based on the copolymers with phase transition temperature, which are polymerized by 2-(dimethylamino)ethyl metharcylate (DMAEMA) and N-isopropylacrylamide (NIPAM). The hydrogels have great adhesion through their own hydrophobicity, where the amino group of the hydrogel forms a hydrogen bond with the substrates, and the ammonium ion forms an electrostatic complexation when the human body’s temperature is higher than the lower critical solution temperature (LCST). The hydrogel adhesive has high viscosity above 37 °C, can maintain the adhesive state, and has reduced adhesion at room temperature, which means that it is easy to remove from the skin. This study demonstrates the potential application of the developed hydrogel adhesives in artificial skin and self-powered biomechanical monitoring system.N,N-Methylenebisacrylamide  and the UV absorber (UV F-22) were purchased from Weihai Huaen Rubber Plastic New Material Co.  Alkaline alumina was used (200–500 mesh).2-(Dimethylamino)ethyl metharcylate  and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone  were purchased from Aladdin Reagents Co. . N-Isopropylacrylamide  was purchased from TCI . The gradient PDMAEMA/PNIPAM hydrogels were fabricated by free radical polymerization under UV irradiation for 4 h and in the presence of a UV adsorber. In brief, the total content of the two monomers in the solution was 30 wt %, and the ratio of DMAEMA to NIPAM was 10:1, such that there was 0.15 wt % MBAA (cross-linker), 0.3 wt % 2959 (photoinitiator) and 0.1 wt % UV absorbers. These were sequentially dissolved into deionized water and the mixed solution was stirred. After standing, the precursor solution was injected into a home-made mold consisting of two glasses and a spacer. Then, then the mold was placed under 360 nm ultraviolet light for 4 h to polymerize. The PDMAEMA/PNIPAM gradient hydrogel was finally obtained by radical polymerization under ultraviolet light.The self-powered hydrogel sensor can be easily integrated by connecting the upper and lower sides of the hydrogel to two copper foil electrodes respectively. During the sensing processes, the hydrogel sensors were attached to the tested solid or skin surface by commercial biaxially oriented polypropylene (BOPP) tape and copper foil tape. The stress and strain sensing performance as well as the large deformation of human body based on the hydrogel sensors were measured.The cross-sectional morphologies of the PDMAEMA/PNIPAM gradient hydrogels were observed using a scanning electron microscope . The element content distribution on the upper and lower surfaces of the gradient hydrogels were analyzed using X-ray photoelectron spectroscopy . The transparency of the receptors was characterized by an ultraviolet and visible spectrophotometer . The mechanical properties were characterized with the strip-like hydrogel using a testing machine (WDW-5T) at tensile speed of 5 mm/min. The peel strength of the hydrogel film was measured with a peeling speed of 20 mm/min using a mechanical testing machine (WDW-5T). The electrochemical measurements were performed on a CHI660 electrochemical workstation . The sensing performance and the output voltage of the receptors regarding external stimulus such as pressure and strain were monitored by a digital source meter .The hydrogels were prepared by free radical polymerization, and the gradient structure was induced by ultraviolet light irradiation in the presence of an absorber . FirstlyThe gradient structure of the hydrogels was characterized by SEM and XPS, respectively. The HD side of the hydrogel has a denser polymer network than the other side, according to the cross-sectional SEM image of the hydrogels a. In addIt is important to manipulate the phase transition temperature of the hydrogel to be close to the temperature of human body. The transition temperature of the hydrogel was tuned by modifying the DMAEAM:NIPAM weight ratio and was measured via turbidity measurements. The mechanical properties of gradient hydrogel membranes were further investigated to ensure that they can be used at a higher temperature than LCST. Their tensile mechanical properties at different temperatures were measured, respectively. As shown in 2 respectively, which is much higher than that at room temperature. This is because the hydrogel loses water at high temperature, and the hydrogen bonds and electrostatic complexation of the hydrogel increase. With the increase of the proportion of DMAEMA monomer, the interaction of hydrogen bonds increase, the average peel strength between the hydrogel and various substrate materials is also increasing. When standing in ambient air for a while or cooling with water to room temperature, the hydrogen bonds between the hydrogel and the base material break, the average peel strength decreases, and the hydrogel is easy to peel. Therefore, no matter the cooling method, the adhesion between the hydrogel at room temperature and the tested substrate surface is poor. Therefore, in addition to its application in skin devices, the PDMAEMA/PNIPAM hydrogels can also be used in other specific scenes such as the various substrates requiring adjustable adhesion. To illustrate the reversible adhesion capacity of gradient hydrogel, we used DMAEMA: NIPAM = 10:1 hydrogel with silicone rubber as the adhesive substrate and carried out 10 repeated peel tests on it. It was found that the interfacial toughness fluctuates slightly and only decreases slightly, as shown in The gradient PDMAEAM/PNIPAM hydrogels can realize the reversible adhesive on various solid surfaces by adjusting the temperature due to their thermal sensitivity. The hydrogel films are adhered to flat or bent insulating substrates, including platinum, copper, silicone rubber, and glasses by sticking and gently pressing a–d. In oF, R and T denote the Faraday constant, gas constant, and temperature respectively, and Ac-C(h) and Ac-C(l) are ions (OH−) concentration on HD side and LD side of gradient hydrogel respectively.It is important to explore the sensing mechanism of the self-powered gradient hydrogels. The self-powered potential of PDMAEMA/PNIPAM gradient hydrogel can be calculated as follows:kth and (k + 1)th) can be expressed as:Ac-(k)C and Ac-C(k + 1) represent the concentrations of OH− at the middle of kth layer and (k + 1)th layer. Therefore, the total built-in potential of gradient hydrogel can be converted intoThe gradient hydrogels can be considered as an aggregate of many ultrathin homogeneous layers, whose density of charged groups increases gradually along the vertical direction . AccordiAc-C(n) and AcC-(1) represent OH− concentration on the upper surface of the n-th ultrathin layer and the bottom surface of the first ultrathin layer respectively. When the sensor is stretched or squeezed, the thickness of each ultrathin layer decreases, leading to a reduction in diffusion distance (− diffuses from the (k + 1)th layer to the kth layer (or from nth layer to 1th layer). Therefore, the ratio of Ac-C(k + 1)/Ac-C(k) and Ac-C(n)/AcC-(1) is decreased, resulting in a decrease in the output voltage of the sensor when it is stretched or pressed.In the formula, distance , and morThe gradient PDMAEMA/PNIPAM hydrogels also can be applied as self-powered ionic sensors as their self-induced potential varies with thickness. When an external pressure is applied to the self-powered hydrogel sensors, its maximum output voltage  gradually decreases along with the reduction of thickness a. The prε) is defined as Sε = |ΔV/Δε|, where Δε is the change of the applied strain. A relatively high strain sensitivity of 2.71 can be obtained under a small strain of less than 40%. When the strain increases to 100%, the strain sensitivity decreases to 1.53. The hydrogel sensor has a wide strain sensing range and can generate repeatable waveforms to respond to 10% to 100% strain , the amino groups of DMAEMA can form hydrogen bonds with the substrate materials, and the ammonium ions form electrostatic complexation. The hydrophobicity also contributes to the adhesion ability of the hydrogels. When the temperature drops below the LCST, the hydrogel becomes easy to separate due to the breaking of hydrogen bonds. Additionally, benefiting from the thickness-dependent potential of gradient hydrogel, the sensors can precisely perceive a tiny variation in pressure and accurately sense tiny changes in strain. The hydrogel sensors can sense the changes of human fingers, wrists, and other joints. This study proves the potential application of self-powered gradient hydrogel in artificial skin and human biological monitoring system.


Gfap) mRNA and allograft inflammatory factor 1 (Aif1/Iba1) mRNA in the denervated oml. We compared the use of single RGs for normalization with the normalization index and found that single RGs yield variable results. In contrast, the normalization index gave stable results. In sum, our study shows that qPCR can yield precise, reliable, and reproducible datasets even under such complex conditions as brain injury or denervation, provided appropriate RGs for the model are used. The algorithm reported here can easily be adapted and transferred to any other brain injury model.Quantitative PCR (qPCR) is a widely used method to study gene expression changes following brain injury. The accuracy of this method depends on the tissue harvested, the time course analyzed and, in particular on the choice of appropriate internal controls, i.e., reference genes (RGs). In the present study we have developed and validated an algorithm for the accurate normalization of qPCR data using laser microdissected tissue from the mouse dentate gyrus after entorhinal denervation at 0, 1, 3, 7, 14 and 28 days postlesion. The expression stabilities of ten candidate RGs were evaluated in the denervated granule cell layer (gcl) and outer molecular layer (oml) of the dentate gyrus. Advanced software algorithms demonstrated differences in stability for single RGs in the two layers at several time points postlesion. In comparison, a normalization index of several stable RGs covered the entire post-lesional time course and showed high stability. Using these RGs, we validated our findings and quantified glial fibrillary acidic protein ( To ensure accurate and reproducible data, qPCR experiments comprise several aspects, including experimental design, sample preparation and data analysis6. The use of reference genes (RGs) is the most common strategy to normalize target gene expression of interest. However, their suitability must be experimentally validated11. Several studies demonstrated that expression levels of some commonly used RGs might vary considerably depending on the specific condition investigated, e.g., traumatic brain injury16. Unfortunately, there is no ideal RG for all experimental conditions and stability of RGs cannot be simply assumed. Under conditions of brain injury, the situation may be even more complex, because gene expression for multiple pathways of injury and repair vary with time following lesion19 as well as with age22. Furthermore, gene expression levels of neurons and glial cells depend on their distance from the lesion23 and, thus, studies using laser microdissected tissues may provide more precise, robust and reproducible results than studies using homogenized larger tissue blocks24.Quantitative polymerase chain reaction (qPCR) has emerged as a standard for precise analysis of quantitative changes in gene expression, especially when only a few target genes are examined or when a small amount of tissue is used28. In this model, transection of the perforant path results in the denervation of the outer molecular layer (oml) of the DG, accompanied by a strong glial reaction30. Candidate RGs were chosen based on earlier entorhinal lesion studies of the rodent brain32 and because of other relevant brain injury studies36. In contrast to previous studies using total hippocampal tissue following entorhinal lesion32, we used laser microdissection to harvest two hippocampal layers, i.e., the granule cell layers, where the denervated granule cells are located and the oml, i.e., the zone of denervation where strong glial reactions have been reported38. Expression stability of ten putative RGs, i.e., actin, beta (Actb), aminolevulinic acid synthase 1 (Alas1), beta-2 microglobulin (B2m), glyceraldehyd-3-phosphate dehydrogenase (Gapdh), hypoxanthine guanine phosphoribosyl transferase (Hprt), phosphoglycerate kinase I (Pgk1), peptidyl propyl isomerase A (Ppia), ribosomal protein L13A (Rpl13a), succinate dehydrogenase complex subunit A (Sdha) and transferrin receptor (Tfrc), were evaluated using commercially available software algorithms, i.e., NormFinder39 and geNorm40, and consensus ranking analysis41. To validate our findings, two prominent glial genes were chosen, i.e., glial fibrillary acidic protein (Gfap), a marker for reactive astrocytes, and allograft inflammatory factor 1 (Aif1)—better known as ionized calcium binding adaptor molecule 1 (Iba1)—a marker for activated microglia cells. Both genes are known to be upregulated after brain injuries and following entorhinal lesion44. Our study showed that single RGs were not sufficiently stable to study the time course of these genes over the entire postlesional time. In contrast, an index of several RGs yielded reliable, reproducible, and accurate results. This index should be used for normalization of qPCR data following entorhinal denervation and similar indexes should be identified using the algorithm described here for other lesion models to provide accurate qPCR data.To demonstrate the usefulness of this combined approach in a proof-of-principle experiment, we investigated the expression stability of candidate RGs in the dentate gyrus (DG) following entorhinal cortex lesion, a classical model for neural reorganization of the brain after injury29 was used to examine the expression stability of putative RGs in the denervated adult mouse DG  and oml of the DG to prove for abundance as well as to examine the variability of selected RGs. Laser microdissection was used to dissect and harvest the two dentate layers Fig. a. RNA inded Fig. b.Figure Hprt and Ppia as the best pair of RGs, whereas geNorm determined Gapdh, Pgk1 and Hprt as the most stable RGs for qPCR normalization. Summarized by RankAggreg, the consensus stability list of RGs for the entire lesion period reads as follows (most stable to least stable): Gapdh–Pgk1–Hprt–Ppia–Sdha–Rpl13a–Actb–Alas1–B2m–Tfrc.Ten putative RGs were tested for their expression stability in the dentate gcl at different time points following entorhinal denervation, i.e., 1, 3, 7, 14 and 28 days. RGs were ranked according to the stability values calculated by NormFinder and by averaged expression using geNorm. RG stability measurements calculated by these algorithms were then used for a consensus ranking analysis employing RankAggreg Table a. CalculAlas1, Hprt, and Tfrc were excluded, see above) was studied in the microdissected dentate oml after entorhinal denervation using the same approach as for the gcl, i.e., using geNorm, NormFinder and a consensus ranking analysis by RankAggreg : Sdha–Ppia–Gapdh–Rpl13a–Actb–Pgk1–B2m.Expression stability of seven putative RGs  against a single candidate RG, (2) against RG indices calculated by geNorm and NormFinder for each lesion time-point, and (3) using the recommended minimal number of RGs for normalization for the entire lesion period based on the consensus ranking analysis. Normalization against a single RG resulted in high variabilities of Gfap mRNA expression levels in the denervated oml  was found. Likewise, using the minimal number of RGs for accurate normalization determined by geNorm, Gfap mRNA expression was also found to be significantly upregulated at 3, 7 and 14 dpl   and 7 dpl , respectively. Finally, using a consensus stability ranking taking all time points and both software algorithms into account, an index of the most stable RGs, i.e., Sdha, Ppia, Gapdh, was used for normalization. Here, a significant upregulation of Aif1 mRNA was found again at 3 dpl and 7 dpl as well as at 14 dpl with corresponding expression levels of 3.3-, 3.1- and 2.4-fold compared to control situation , which found that Actb was significantly upregulated at 2 dpl in the deafferented hippocampus, while at 7 and 15 dpl no apparent change of expression relative to control was detectable16. Interestingly, only Ppia (Cyclophilin A) demonstrated a relatively high expression stability at all lesion time points  in this study. In our study, Ppia was recognized as a suitable RG in the denervated oml, too. In addition, various studies analyzed Ppia in other commonly used brain injury models and identified it as being one of the most stable RGs64. In contrast, B2m was recognized as a less stable RG in several studies63, but was postulated also as suitable RG in a few research papers65. In our study, B2m was identified as the least stable RG in the denervated oml at all time points analyzed following lesion. In contrary, Sdha was identified as the most stable RG in the oml throughout all time points after denervation. This is in accordance with previous brain injury studies, where Sdha was used as RG for accurate qPCR normalization65.Initially, the expression of up to ten selected RGs was evaluated in two DG layers, i.e., the gcl and oml, to examine for abundance and variability of RGs. Of note, a few putative RGs had to be excluded from further validation analysis in the denervated oml, since they showed insufficient amplification because of low abundance expression, which could have influenced accurate qPCR quantification. Therefore, ten candidate RGs for the gcl and seven RGs for the oml were investigated for their expression stability after entorhinal denervation. Validation of RGs was performed at five individual time points after lesion, i.e., 1, 3, 7, 14 and 28 dpl, compared to control situation. Gfap and Aif1/Iba1, respectively. Both genes are upregulated in the denervated zone and reflect the strong glial response to entorhinal denervation66. Using immunofluorescence labeling for AIF1/IBA1 and GFAP, a major upregulation of these proteins in microglial cells as well as astrocytes, respectively, could also been seen at the protein level at 3 and 7 dpl in the denervated oml29. In previous studies, Gfap mRNA levels were measured in hippocampal samples using dot plot hybridization analysis67 or using laser microdissection in combination with qPCR using a single RG for qPCR normalization24. We now extended these earlier studies by analyzing the effect of different qPCR normalization strategies: target expression levels of Gfap and Aif1 mRNA were normalized in the denervated oml (1) against single candidate RGs, (2) against RG indices calculated by geNorm or NormFinder for each specific time-point following denervation, and (3) using the minimal number of RGs calculated for accurate qPCR normalization based on a consensus stability ranking for the entire lesion period and across the software algorithms used. Normalization against a single RG demonstrated a relatively high variation of mRNA expression levels for Gfap and Aif1 in the denervated oml. Significant upregulation of Gfap mRNA expression was found at 3, 7, 14 and 28 dpl depending on the RG that was used for normalization. Except for Pgk1, maximal upregulation of Gfap mRNA was detected at 7 dpl with expression levels varying considerably from 6.5- fold using B2m to 19.8- fold using Rpl13a. Similarly, a significant upregulation of Aif1 mRNA expression was found either at 3, 7 or 14 dpl depending on the RG used for qPCR normalization. Maximal upregulation of Aif1 mRNA was identified at 3 dpl using Gapdh, Pgk1, Rpl13a or Sdha. In contrast, normalization against Actb or B2m resulted in no significant expression changes of Aif1 mRNA. Of note, B2m was found to be the least stable RG in the oml at all time points analyzed following denervation. Similarly, Actb should also not be considered at earlier time points after denervation, but might be more suitable at later time points, which was also shown in the study of Harris and colleagues (2009). Using an index of suitable RGs according to the different software approaches by NormFinder and geNorm, a significant upregulation of Gfap mRNA at 3, 7 and 14 dpl and for Aif mRNA at 3 and 7 dpl was found. Importantly, a maximal upregulation of Aif1 mRNA was observed at 3 dpl, whereas maximal upregulation of Gfap mRNA upregulation was found at 7 dpl, which in both cases returned to lower levels at later time points after injury. Finally, qPCR normalization was performed using an index of the three most stable RGs, i.e., Sdha, Ppia and Gapdh, calculated based on a consensus stability ranking by including all lesion time-points and both software algorithms used. As a result, a significant upregulation of Aif1 mRNA and Gfap mRNA was found for both transcripts at 3, 7 and 14 dpl. Again, maximal upregulation of Aif mRNA was observed earlier (3 dpl) compared to Gfap mRNA (7 dpl). These results are in line with the early response of microglia cells, followed by the later reaction of astrocytes in rodents following entorhinal lesion66. Differences in the detailed time course of Gfap mRNA expression might be explained by anatomical species variations between mouse and rat or by distinct lesion techniques26. Our results demonstrate that normalization against non-validated or relatively non-stable RGs can lead to inconsistencies in qPCR expression analysis and, consequently, might lead to misinterpretation of real biological data. In contrast, normalization against an index of validated RGs determined by advanced software algorithms yields more accurate data. In addition, the precise harvesting of tissue, e.g., by using laser microdissection, helps to identify local changes after brain injury, which is of the essence to understand the sequence of events following damage. Based on our results, a normalization index with a minimal number of suitable RGs is sufficient for accurate qPCR quantification of target genes following entorhinal denervation. For a more precise analysis, we would recommend using time-point specific reference gene sets. However, the usage of a more broader normalization index of RGs calculated by consensus stability ranking for an entire lesion period is also highly appropriate and, thus, might be used in forthcoming studies, where more lesion time-points across different subregions are analyzed.As proof-of-principle, we used the RGs found to be suitable for accurate qPCR normalization after denervation to analyze the expression of two mRNAs of astrocytes and microglia, i.e., Map2 mRNA in granule cells. Microdissection of this neuronal layer is an excellent tool to harvest tissue enriched in granule cell mRNA, since granule cells are densely packed in this layer and greatly outnumber other neurons and glial cells. However, in contrast to glial cells, which react strongly to the degeneration of axon terminals and axons in the outer molecular layer, granule cells show only a very weak response. In fact, earlier studies performed in rats46 reported only very mild changes, which were an order of magnitude weaker than the changes found in glia and which were close or even under the threshold of detection using classical methods. Using laser microdissection and qPCR with a reference gene index established in the present study, we could, however, detect a mild upregulation of Map2 mRNA in mouse granule cells around day 3 to 7. This demonstrates that the algorithm proposed in this study also works with neurons and most likely any other cell type and allows for the identification of an index of stable and reliable reference genes.To demonstrate the usefulness of our algorithm for the detection of neuronal genes, we also analyzed changes in In conclusion, we have shown here using the well-established and thoroughly studied entorhinal cortex lesion model, that tissue from different time points after lesion harvested with laser microdissection and analyzed with qPCR yields precise, reliable, and reproducible quantitative datasets. To achieve this precision, it was of the essence to identify an index of RGs that is (i) appropriate for the model and (ii) covers the entire time course postlesion. The algorithm reported here can easily be adapted and thus transferred to any other brain injury model, making qPCR data in the field of brain injury and regeneration more precise and reproducible.For experimental analysis, adult male mice  were used. Mice could survive 1, 3, 7, 14 or 28 days after entorhinal denervation. Experiments were carefully designed to examine control and lesioned animals of similar age.Animal care and experimental procedures were performed in agreement with the German law on the use of laboratory animals  and approved by Regierungspräsidium Darmstadt . The animal experiments described in this study were conducted in accordance with ARRIVE guidelines.29. Correct placement of the wire knife cut was verified on horizontal brain sections  containing the lesion site and parts of the temporal dentate gyrus (DG). In addition, entorhinal denervation was verified on frontal hippocampal sections (25 µm) using Fluoro-Jade C (HistoChem Inc.) at early time points post lesion to monitor the appearance of degeneration products68 and counterstained with Hoechst 33242 (Invitrogen).Unilateral transection of the perforant path was performed using a wire knife  as described previouslyMice were deeply anesthetized with an overdose of pentobarbital (300 mg/kg body weight) and transcardially perfused with 0.9% sodium chloride (NaCl) followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (pH 7.4). Brains were removed, post-fixed for 24 h in 4% PFA and sectioned in the coronal plane (40 µm) using a vibratome . Free-floating sections were incubated in a blocking buffer containing 0.5% Triton X-100 and 5% bovine serum albumin (BSA) in 0.05 M Tris-buffered saline (TBS) for 30 min at room temperature followed by incubation in the primary antibody (diluted in 0.1% Triton X-100 and 1% BSA in 0.05 M TBS) overnight at 4 °C. The following primary antibodies were used: rabbit anti-GFAP , mouse anti-NeuN  and anti-AIF1/IBA1 . After several washes, sections were incubated with the appropriate secondary Alexa-conjugated antibodies  for several hours at room temperature and finally mounted in DAKO Fluorescent Mounting Medium (Dako).Figures were prepared digitally using commercially available graphics software (Photoshop Adobe Inc.). Fluorescent images were acquired using a digital camera  or confocal microscopy . Single fluorescent images of the same section were digitally superimposed. The contrast, brightness and sharpness of images were adjusted as needed for each section. No additional image alteration was performed.For layer-specific analysis of the granule cell layer (gcl) and outer molecular layer (oml) of the DG, prepared brains were embedded in tissue freezing medium and immediately flash-frozen in − 70 °C isopentane cooled by dry ice for 2 min. Until further processing, brains were transferred and stored at − 80 °C. For laser microdissection, 16 µm thin brain sections were cut using a cryostat (Leica Biosystems) and mounted on polyethylene naphthalene (PEN) membrane slides (Leica Microsystems). Sections were dried shortly at room temperature (RT), fixed in − 20 °C cold 75% and 100% ethanol (AppliChem) and stored at − 80 °C until further processing. Before laser microdissection, sections were thawed and stained quickly with 1% cresyl violet staining solution (Sigma-Aldrich) at RT and briefly dehydrated in 75% and 100% ethanol. Using a Leica LMD6500 system (Leica Microsystems), defined tissue portions of the gcl and oml of the DG from the same brain sections were collected separately in 50 µl lysis buffer  with ß-mercaptoethanol. Tubes were refilled to 350 µl with lysis buffer, vortexed for 30 s and transferred immediately to − 80 °C until further processing. Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s recommendations. RNA integrity was assessed using the Agilent 2100 Bioanalyzer system and Agilent RNA 6000 Pico Kit (Agilent Technologies). Only high RNA quality  samples were used for further processing.Actb), aminolevulinic acid synthase 1 (Alas1), beta-2 microglobulin (B2m), glyceraldehyd-3-phosphate dehydrogenase (Gapdh), hypoxanthine guanine phosphoribosyl transferase (Hprt), phosphoglycerate kinase I (Pgk1), peptidyl propyl isomerase A (Ppia), ribosomal protein L13A (Rpl13a), succinate dehydrogenase complex subunit A (Sdha) and transferrin receptor (Tfrc)  following the manufacturer’s recommendations. Quantitative PCR (qPCR) was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). qPCR conditions were carried out using EXPRESS SYBR GreenER qPCR SuperMix with Premixed Rox (Invitrogen) and a final primer concentration of 500 nM. Primers were designed to be intron-spanning to exclude amplification of genomic DNA. Ten potential reference genes (RGs) were selected for analysis: actin, beta , which is the average pairwise variation of a single gene to all other putative RGs and is the result of a stepwise exclusion of the least stable gene within the panel of RGs. In addition, to reach an unbiased consensus for both software algorithms, a comprehensive ranking analysis was performed using RankAggreg41. Minimum number of RGs for accurate normalization was calculated based on optimal gene rank lists by pairwise variation between two sequential normalization factors containing an increasing number of genes40. Based on this study, 0.15 was set as a cut-off, below the inclusion of an additional RG was not required for proper normalization. Statistical analysis of qPCR data was performed using GraphPad Prism 6 software. For one-way ANOVA, multiple comparisons between individual time-points and control were used in combination with Dunnett’s multiple comparisons test. P-values < 0.05 were considered statistically significant.To evaluate the gene expression stability of candidate RGs, two different software algorithms, i.e., NormFinderSupplementary Information 1.Supplementary Information 2.


The present study aimed to evaluate the effectiveness of the Brainballs on the physical fitness of 2nd-grade students at a primary school in Vietnam during and eight months after the experiment.The study included 55 pupils (23 boys and 32 girls) aged seven years. The study design was a pedagogical experiment with a parallel-group technique, including experimental and control groups. The examination was carried out in 2019/2020 in three terms pre- (September 2019), post- (January 2020), and follow-up (September 2020). Physical fitness was tested with the use of the International Physical Fitness Test. The Brainball program, conducted twice a week for 35 minutes, combined physical education (PE) with subject-related content, utilizing 100 balls with painted letters, numbers, and signs.p = 0.0044), toe touch (p = 0.0137), standing long jump (p = 0.0076), 4 × 10 m sprint (p = 0.0333), hand strength (p = 0.0233).Results show that the fitness level was not increased significantly after 20 weeks of the intervention program, neither in experimental nor control groups. However, it significantly improved eight months later at the follow-up examination. The analysis of covariance indicated that pupils from the experimental group improved significantly on most physical fitness as compared to the control group, specifically on the following tests: 50-meter running (These results have shown long-term positive effects of the use of “Brainball” educational balls in physical education classes on the physical fitness development of students, especially in the qualities of speed, strength, and flexibility. Evidence  proves tFor children and adolescents, PA includes games, sports, recreation, physical education (PE), or planned exercise as part of the family, school, and community activities . HoweverIn an effort to find a new teaching method to improve teaching and learning for students, researchers at the Wroclaw University of Health and Sports Science, Poland, have created an active teaching method called Eduball/Brainball. The primary teaching method of the program is to use games and exercises designed with educational balls to integrate the contents of other subjects into physical education classes , 17. AftThe idea of the educational balls was coined in 2002 in the Department of Team Sports Games at the Wroclaw University of Health and Sport Sciences, Poland. The first name of educational balls was “Edubal.” After 10 years of experience and research with Edubals, the set was modified and the next edition of educational balls, called “Eduball,” was prepared. In 2018, the English edition of education balls called “Brainball” started; although the name is different, the idea of Edubal/Eduball/Brainball is the same; children learn while playing! .*), division (:), greater than (>), less than (<), parentheses , and the at sign (@)]  . The setign (@)] . The numign (@)] . The gamign (@)] , 32.Eduball/Brainball has been implemented in the official list of teaching aids for elementary schools in Poland. They were accredited and approved by the Polish National Ministry of Education. Eduball/Brainball has also been launched and confirmed its benefits in Germany, Portugal, Finland, Greece, the USA, Singapore, and Taiwan (China) . HoweverThe research sample was students from two second-grade classes in a primary school in the center of An Giang province, a province located in the Mekong Delta region of Southern Vietnam. A total of 55 students  participated in the study. The study was conducted during the 2019–2020 academic year. The study design included pedagogical experiments using parallel grouping techniques under natural conditions. Participants were randomly divided into a control group of 27 students (11 boys and 16 girls) and an experimental group of 28 students (12 boys and 16 girls). The teaching process of both groups  was conducted according to the same curriculum prescribed by the Ministry of Education and Training of Vietnam. The only difference is the introduction of Brainball into the teaching and learning in the experimental group. All participants' parents and guardians signed informed consent for their children to participate in this study. The study was approved by the local Ethics Committee for Research Involving Human Subjects . It was conducted according to the principles of the Declaration of Helsinki.The experimental factor was a PE program combined with Brainball games and exercises . The expIn the control group, physical education classes also took place twice a week for 35 min and were conducted using traditional styles . The same PE teacher with 10 years of experience, taught physical education in both groups . Due to the experiment the teacher was trained in the Brainball method to organize and perform games and exercises with these balls.The International Physical Fitness Test  was usedAt the command “on your marks,” the pupil doing the exercise stands still in front of the starting line with one leg put forward . Then, at the “start” signal, he runs to the finish as quickly as possible. Time is measured with an accuracy of 0.1 s.The subject stands with legs extended; naturally, toes are placed close to the boundary line; when jumping and landing, both feet do it simultaneously. The jump length is measured from the setline (beam) to the nearest footstep left by the jumper's heel. The result is the longest distance jumped, the best of two attempts.The subject stands with feet shoulder-width apart. The dominant hand holds the dynamometer toward the palm and extends straight along the body. The subject performed the exercise by reducing the dynamometer to maximum power. The result is the highest of two attempts.The task is to remain as long as possible, hanging with arms bent in elbow joints. Upon starting the test, the person doing the exercise holds the bar with fingers directed downwards and the thumb from the bottom upwards, at the shoulders' breadth, so that his chin would be above the bar. The test starts when the person doing the exercise hangs on the bar unaided and ends when his eyes go below the bar. Time is measured in 0.1-s units.The subject lies supine on a mattress, knees bent, hands clasped on the neck, and performs sit-ups, feet held firmly by an assistant. The result is the exact number of sit-ups in 30 s.The subject stands with one foot forward (standing start) in front of the starting line. On signal, the subject runs to the finish line to pick up a block, runs back, and places the block behind the start line. The subject then runs back to the finish line, picks up a second block, and runs back to put it behind the start line. If the block is thrown and not placed behind the starting line, the test is considered invalid and must be repeated. Time is measured with an accuracy of 0.1 s.The subject was not wearing shoes, stood on a stool or bench, toes placed close to the edge of the stool, feet together, and knees straight. From this position, the person doing the exercise bends forward with a continuous movement to reach the furthest with his fingers. Such a position of a maximum bend must be kept for 2 s. If the person doing the exercise reaches the level he is standing on while bending with a continuous movement, he scores 0. He scores a plus point for every centimeter below the surface of the stool. Otherwise, he scores a minus point for every centimeter above the surface of the stool. The test is invalid if, during bending, the legs are bent in knee joints. Any vigorous movements during bends are not permitted, either. The result is the best of two attempts.All measurements were taken in September 2019 (marking the beginning of academic year), and in January 2020 . The final, third examination took place in September 2020  to estimate the long-term impact. Technical researchers conducted fitness tests on the training ground during physical education classes. Measurements were carried out in a natural environment with the help of teachers and students. Additionally, principals, teachers, and parents approved information on testing procedures before testing.t-tests for the dependent variables were used to compare the differences in the mean parameters of the tests performed between the experimental and control groups. Next, to determine the statistically significant differences between the experimental and control groups after 1 year of study, an analysis of variance (ANOVA) was performed. Partial eta squared (ηp2) was used to quantify the effect size   was used for statistical analysis. The main dependent variables were the scores on the physical fitness test obtained by testing students in the control and experimental groups. First, the Shapiro-Wilk test confirmed the normal distribution of the physical fitness test. Then, the students'  = 0.14) . Newmanap < 0.001.The results in F = 5.72, p = 0.004] and toe touch , standing long jump , 4 × 10-meter sprint , and hand strength [F = 3.90, p = 0.023; A repeated-measures analysis of variance ANOVA (2 × 2) was performed to compare the effectiveness of the Brainballs program on pupil physical fitness. The analysis showed that after 1 year of study, there were significant differences in the level of physical fitness improvement between the groups. Pupils in the experimental group were significantly better than those in the control group in most tests, especially the 50-meter run [p = 0.004), toe touch (p = 0.014), standing long jump (p = 0.008), 4 × 10 m sprint (p = 0.033), and hand strength (p = 0.023) tests. These findings indicated that the Brainball program did not positively affect the pupils' physical fitness development in the experimental period, however, it showed progress in the follow-up, which may indicated long-term positive effects on the pupils' physical fitness development.The main objective of this study was to evaluate the effectiveness of Brainball program on the physical fitness of 7-year-old children in a primary school in Vietnam. The Brainball program is an innovative, comprehensive educational program that encourages and provides opportunities for intensive activities to improve health-related fitness and motor performance skills and enhance achievement learning to improve academic performance in comprehensive content. The study showed that after 20 weeks of the experiment, there was no significant difference in the level of physical development of the pupils in both groups . However, after a year of study, there were significant differences between the two groups. The improvement in physical fitness of the pupils in the experimental group was significantly better than that of the pupils in the control group, especially in 50-meter running . Compared with previous studies, when educational balls were applied in Poland  in case of Vietnamese intervention the time duration (2 times a week 35 min) might have been an issue. Exciting physical activities with colorful balls  could help pupils actively participate in movement, improve coordination, and develop motor skills, but did not impact level of fitness so significantly as the operational time was too short for pupils to absorb and develop fitness. Explanation may come from Rink , who staThe follow-up test results showed that after 1 year of study, the physical fitness of the pupils in the experimental group was significantly improved compared to the pupils in the control group, especially in terms of speed, strength, and flexibility. This suggests an association between the use of Brainballs in physical education classes and the physical development of pupils in the experimental group. Games and exercises with Brainball can be the cause of this relationship. Exciting activities with educational balls have made the class lively and attractive, students actively participate in and acquire knowledge . All theResearchers have previously demonstrated that the development of physical fitness is not only influenced by physical activities and sports, but also by factors such as genetics, environment ; socioecThere are limitations to our study. Firstly, this is the first study to apply for the Brainballs program in physical education classes for students in Vietnam. Teachers and students are very interested in this new method of teaching and learning, but sometimes they are confused and unfamiliar with how to organize and perform exercises. Secondly, the study sample is relatively small, so it is difficult to generalize the research results. Despite its limitations, the study has many advantages. The study used an experimental pedagogical design with two parallel groups conducted in a natural environment. The students voluntarily participated in this study and received special attention from principals, teachers, and parents. During the implementation, classroom and physical education teachers coordinated rhythmically in the content of the curriculum, which is a new thing in the Vietnamese education system. The findings provide further insight into the effectiveness of using educational balls to promote healthy physical development in Vietnamese 7-year-olds' physical education classes. As for the pupils, they had the opportunity to experience new ways of learning and participate in exciting sports activities with Brainballs. This helped increase their interest while learning and fostering the absorption of knowledge. In addition, the results of this study will be the premise for further studies with a broader and deeper scope of research to precisely evaluate the impact of Brainball on the physical fitness development of students.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Resolution of the Senate Committee on Ethics of Scientific Research at the Wroclaw University of Health and Sport Sciences in Wroclaw. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.VP, AR, and MB developed ideas, managed the project, provided critical scholarly guidance for the drafting and interpretation of the manuscript, and critically revised the intellectual content of the manuscript. VP, SW, IC, and MB researched background literature, analyzed data, and wrote the manuscript. All authors approved the final version of the manuscript, ensured the accuracy of the work, and agreed to take responsibility for all aspects of the work.